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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Research Project #421196

Research Project: Biology and Control of Human Pathogens on Fresh Produce

Location: Produce Safety and Microbiology Research

2011 Annual Report

1a. Objectives (from AD-416)
Objective 1: Identify and chacterize the microbial genes that are involved in the attachment, colonization and survival of enteric pathogens on produce. Objective 2: Determine the genetic and biochemical factors in plants that effect the attachment, growth and survival of human pathogens in/on plants. Objective 3: Assess the role of other microflora and aerosols in survival and transmission of enteric pathogens in agricultural environments. Objective 4: Develop methods for the detection of enteric viral and bacterial pathogens from produce and soil.

1b. Approach (from AD-416)
Plant-microbe model systems in combination population studies, molecular methods, genomics, and microscopy, will be used to investigate the interaction of human pathogens with plants and plant-associated bacteria, as well as well as to develop improved methods for detection of human pathogens on produce. Replacing 5325-42000-044-00D 03/2011.

3. Progress Report
In the light of the large outbreak of enteroaggregative E. coli O104:H4 in Europe in 2011, frozen samples from a survey of pathogenic E. coli in the Salinas region, CA, are being re-screened for specific E. coli serotypes (O104 and O55), as well as for enteroaggregative and extra-intestinal pathogenic E. coli. As non-O157:H7 shigatoxin-producing E. coli (STEC) are emerging in the US, we began comparative genomics of STEC O145, a strain of which caused an outbreak linked to lettuce from a major produce-growing region of the US. Additionally, a method was developed for high throughput screening of fitness traits in numerous STEC strains isolated from the Salinas Valley in order to improve our understanding of their epidemiology in agricultural habitats, including produce. We previously demonstrated large inter- and intra-strain variations in the production of curli fimbriae in E. coli O157:H7, and difference in stress tolerance among curli variants of the pathogen. We have shown that this variation is likely due to a regulatory switch, suggesting a molecular mechanism in E. coli O157:H7 that generates variants to ensure the survival of the pathogen population under various stress conditions. The genome instability of E. coli O157:H7 was measured after desiccation and acid treatments for comparison to genomic instability observed with starvation, UV and high temperature. We investigated bacterial communities in biofilms that include E. coli O157:H7 and showed that the pathogen is a strong competitor and biofilm producer on abiotic surfaces and leaf tissue when in field water (collected in the Salinas Valley) containing simple nutrients. Survival of E. coli O157:H7 in naturally contaminated soil was monitored on particles dispersed in a wind tunnel, then applied onto lettuce leaves or seedlings. Additionally, the relationship between the size of particles and the level of natural contamination in soil samples was determined. Also, the survival of outbreak strains was monitored in moist soil microcosms under drying conditions that simulate those leading to aerosolization. Regarding our work with Salmonella, we determined levels of resistance to ultraviolet and oxidative stress in 34 different strains of Salmonella of various serotypes and serogroups originating from meat and produce. Nutrient utilization profiles of Salmonella strains were determined using the Biolog system. Studies on molecular detection of Salmonella has continued with testing of various combinations of real time PCR primers and probes with enrichment cultures in addition to immunological methods of detection of various serogroups of Salmonella when co-cultured under standard enrichment conditions. We have extended a survey for the prevalence of Salmonella in California by testing 1000 samples from various animals, and determined the antibiotic resistance of isolates. Concerning our studies of norovirus biology on produce, we have shown that virus binding to lettuce or raspberries was enhanced under acidic conditions mainly due to the interaction of electric charges, suggesting that acidic wash should be avoided for the removal of norovirus from contaminated produce.

4. Accomplishments
1. Cell individuality in E. coli O157:H7 with relevance to its epidemiology. Curli fimbriae are important in host and plant colonization, biofilm formation, and induction of inflammatory response. ARS researchers in Albany, CA, observed large E. coli O157:H7inter- and intra-strain variations in the production of curli fimbriae. Curli variants derived from the same pathogenic strain displayed marked differences in their response to environmental and host-related stresses. Whereas, curli-producing variants were more fit under nutrient limitation, curli-deficient variants were more resistant to acidic pH such as that in the human stomach. DNA sequence analysis indicated that a genetic switch may underlie this phenomenon. These results provide insight into the evolution of the pathogen and are critical to understanding the survival strategies of the pathogen during its cycling between host and nonhost environments, and its epidemiology.

2. Aerosol transport of bacteria from concentrated animal feeding operations. Aerosols have been suspected to transport food pathogens and contaminate fruits and vegetables grown in close proximity of concentrated animal feeding operations. However, studies investigating such transport are scarce, and science-based guidelines for the produce industry regarding the safe distance between produce fields and processing plants, and animal operations are lacking. ARS researchers in Albany, CA, used gene sequencing methods to characterize bacterial communities in manure and air samples collected from dairies in Central and Coastal valleys of California. Whereas known enteric pathogens were not detected in aerosol samples, bacterial species originating from cows at the dairy operation in the vicinity were identified. Thus, bacteria specific to each dairy may be used as tracers for detecting the source of pathogens that are transported to produce or fruit crops from feedlots or dairies. This suggests a new strategy for trace back epidemiological investigations by regulatory agencies and for quality testing by the produce industry.